1. Field of the Invention
The present invention relates generally to the fields of molecular biology and molecular structure of Cryptococcus neoformans. More specifically, the present invention relates to cloning, sequencing and expression of a gene encoding an enzyme which de-O-acetylates glucuronoxylomannan of Cryptococcus neoformans.
2. Description of the Related Art
Cryptococcus neoformans is an encapsulated yeast that exists in two varieties. C. neoformans neoformans has been isolated from pigeon droppings and is found worldwide in temperate climates whereas C. neoformans gattii has been associated with eucalyptus trees and is found in tropical or subtropical regions (13). Both varieties are pathogenic to humans and can produce fatal infections of cryptococcal meningitis (13). The yeast is most commonly pathogenic in immunosuppressed individuals, particularly those with advanced AIDS (8,49). C. neoformans has four distinct capsular serotypes, A through D, which are characterized by unique chemical compositions. (33). The serotype used exclusively for this study is serotype A, which comes from C. neoformans var. neoformans. This serotype is the cause of most cases of cryptococcal meningitis found in AIDS patients (8,49).
Cryptococcus neoformans cells have very large capsules (FIG. 1), which are composed of the polysaccharide, glucuronoxylomannan (GXM). Serotype A GXM has an .alpha.-1,3-D-mannose backbone with one .beta.-D-glucuronide and two .beta.-D-xyloside sugars per each trimer of mannose (FIG. 2) (14). The backbone is also O-acetylated, ranging from approximately 3-16.5%(33). It has been shown that the O-acetylation forms part of the antigenic epitope for some monoclonal antibodies (MAbs) (46). These monoclonal antibodies can, therefore, be used for determining the presence and degree of O-acetylation on glucuronoxylomannan through ELISA antibody capture assays.
The capsule has been determined to be the single most important virulence factor for the pathogenicity of C. neoformans (33). Acapsular mutant strains produced by several laboratories have been found to be avirulent (12,16,35,38). Glucuronoxylomannan has been shown to affect host resistance in a number of ways including, but not limited to inhibition of phagocytosis (10,11,37), suppression of lymphocyte responses and proliferation (9,43), induction of T-cell dependent and independent immunologic tolerance (36,44,53), and even enhancement of HIV-1 infectivity in vivo (47). Treatment consists of the antimycotic agents, amphotericin B and flucytosine, or the azoles, ketoconazole and fluconazole (45). These treatments are often complicated by existing infections and their treatments as well as having some very severe side effects. The disease presents challenges on many fronts to the medical community.
Another challenge is the high viscosity caused by circulating glucuronoxylomannan, and perhaps encapsulated yeast cells, which are thought by some to lead to cerebral edema (23-27,39,40). The edema is characterized by increased intracranial pressure and has not been uniformly amenable to surgical intervention. It can evolve rapidly and be fatal. The soluble glucuronoxylomannan also presents a problem in that it is not cleared from the circulation and tissues of the host very efficiently (32a, 32b. 32c and 32d).
Prior to the advent of antibiotic and antimycotic agents, investigators experimented with enzymes that could degrade capsular polysaccharides. The first study of this type involved the use of a bacterium to degrade the polysaccharide capsule of Type III Streptococcus pneumoniae (4,15). The bacterium was isolated from a cranberry bog in New Jersey. Several studies followed and were expanded into in vivo studies with mice and rabbits. Enzymes were found to be effective in protection against lethal injections as well as in a curative manner when infections had been firmly established prior to treatment (5,51,52). A glucuronoxylomannan-hydrolase was discovered in a similar manner by Gadebusch in 1960. Soil samples tested for enzymatic activity led to isolation of a Gram-negative rod, designated Alcaligenes sp. S-3723, which completely degraded the capsule of C. neoformans (17-20). At the time of the Gadebusch report, the composition and structure of GXM was incompletely and sometimes erroneously understood. In retrospect, the Gadebusch was probably a mixture of two or more unidentified and uncharacterized enzymes. The enzyme cocktail was tested in vivo on mice infected with C. neoformans. The ET.sub.50 increased from 18 days for mice with no treatment to 47 days for enzyme-treated mice.
Gadebusch's findings on this enzyme were published in 1960 and 1961 (17-20). Amphotericin B was gaining acceptance as a lifesaving treatment for cryptococcal meningitis and the disease was not very prevalent at that time. Molecular cloning had yet not been conceived, making the use of enzymic treatments tedious and costly, as enzyme had to be purified through a lengthy process from native bacteria. Today, C. neoformans infects 5-10% of AIDS patients in the U.S. and is the most common life threatening opportunistic fungal infection in AIDS (34). Antimycotic treatments prolong survival of these patients, but are ineffective against the cerebral edema and have little impact on high serum titers of antigen. Enzyme treatment may be the answer to this lingering problem.
The prior art is deficient in the lack of identification of specific GXM-cleaving enzymes and the lack of a gene encoding an enzyme that modifies the structure of the capsular polysaccharide of C. neoformans. Further, the prior art is deficient in the lack of means to block the deleterious activities of GXM that occur during the course of cryptococcosis. The present invention fulfills this long-standing need and desire in the art.